Of the greater than 6 million fractures occurring yearly in the US, up to 20% will result in nonunion or delayed union, thereby requiring intervention for bone regeneration. Mesenchymal stem/stromal cells (MSCs) are an attractive cell source for cell-based therapies of bone healing because of their osteogenic potential and robust secretion of proangiogenic trophic factors. Culture dimensionality has a profound impact on a myriad of cell functions. Compared to dissociated MSCs, our recent data demonstrate that MSC spheroids secrete 100- fold higher levels of angiogenic factors and better resist apoptosis while maintaining osteogenic potential. Spheroid formation is a competition between cohesion and adhesion, and optimizing this balance through the entrapment in engineered biomaterials provides an exciting opportunity to instruct the regenerative potential of MSCs after transplantation. Hydrogel properties such as adhesivity, stiffness, and degradation influence the function of entrapped cells and resulting tissue formation. Alginate is a highly cytocompatible natural polymer that is amenable to control of initial mechanical properties through composition and crosslinking, as well as adhesivity by covalently coupling peptide sequences such as Arg-Gly-Asp (RGD) to the polymer backbone that bind cellular receptors. Thus, alginate hydrogels represent an ideal tool to probe the role of substrate properties on spheroid function. Our central hypothesis is that the therapeutic potential of MSC spheroids for bone regeneration can be enhanced using alginate hydrogels with engineered biophysical properties. Aim 1. Does adhesion ligand density within alginate hydrogels affect the survival, proangiogenic, and osteogenic potential of entrapped MSC spheroids? We will synthesize alginate hydrogels with varying densities of RGD. The influence of increased adhesion versus cohesion on spheroid function will be determined. Aim 2. Do hydrogel biomechanical properties influence the functional response of entrapped MSC spheroids? Using composite hydrogels with distinct biophysical properties, we will examine the role of substrate stiffness and degradation on survival, proangiogenic and osteogenic potential of entrapped MSC spheroids. Aim 3. Can MSC spheroids transplanted in RGD-modified hydrogels with optimized biophysical properties accelerate bone formation in a critical-sized calvarial bone defect? We will characterize the capacity of MSC spheroids transplanted in RGD-modified alginate hydrogels to accelerate bone repair in an orthotopic defect compared to dissociated MSCs. The role of implanted cells, as well as quality of bone formation will be assessed using noninvasive imaging modalities. The proposed research is innovative because it exploits the balance of cellular aggregation versus adhesion to drive cell fate using an injectable, biodegradable hydrogel to potentiate the reparative potential of MSCs. This research will provide a new approach to drive bone formation in nonhealing or slow healing bone fractures, and the strategies have potential in enhancing the efficacy of materials-based therapies for tissue repair.